Higher levels of physical activity are associated with reduced tethering and migration of pro-inflammatory monocytes in males with central obesity.

Despite evidence that monocyte migration is accentuated by central adiposity, the impact of physical activity (PA) and exercise, particularly in the post-prandial state, on limiting migration are not established. We hypothesised that PA and a single bout of walking exercise would be associated with reduced ex vivo monocyte tethering and migration in middleaged males with central obesity (CO). Objective levels of PA were measured for 7 days in lean males (LE, N=12, mean (SD) age 39 (10) years, waist circumference 81.0 (6.3) cm) and males with CO (N=12, mean (SD) age 40 (9) years, waist circumference 115.3 (13.9) cm), followed by donation of a fasted blood sample. On the same day, CO undertook a bout of walking exercise, before donation of a second fasted blood sample. An ex vivo assay, coupled to flow cytometry, determined tethering and migration of classical, intermediate, and non-classical monocytes. C-C and CXC chemokine receptor (CCR2, CCR5 and CX3CR1) expression were also determined on total and classical monocytes. Monocyte subsets (total, classical, intermediate and CCR2+ monocytes), metabolic (glucose and lipids) and inflammatory (C-reactive protein) markers were greater in CO vs. LE (lower highdensity lipoprotein); however, adjustments for PA mitigated group differences for glucose, lipids, and monocyte subsets. Ex vivo tethering and migration (absolute and relative) of most monocyte subsets was greater in CO vs LE. Relative monocyte tethering and migration was largely not influenced by PA; however, higher PA was associated with reduced absolute migration and tethering of CD16 expressing monocytes in CO. Prior walking had no impact on these variables. These results highlight that regular PA, not single exercise bouts may limit the migration of pro-inflammatory monocytes in CO. These changes may relate to physiological parameters in blood (i.e. number of cells and their adhesion), rather than differences in chemokine receptor expression.

The effects of a single night of complete and partial sleep deprivation on physical and cognitive performance: A Bayesian analysis.

This study investigated the effects of complete and partial sleep deprivation on multiple aspects of athletic performance. Ten males completed a cognitive function test, maximal handgrip strength, countermovement jump (CMJ) and a 15 min all out cycling test to assess aerobic performance. These tests were performed following 3 different sleep conditions; normal sleep (CON), a 4 hr sleep opportunity (PART) and complete sleep deprivation (DEP). Data were analysed using a Bayesian multi-level regression model to provide probabilities of impairment (p = %). Aerobic performance, CMJ and handgrip strength were impaired by 11.4% (p = 100%), 10.9% (p = 100%) and 6% (p = 97%) following DEP, while aerobic performance and CMJ were highly likely impaired by 4.1% (p = 90%) and 5.2% (p = 94%) following PART. Cognitive reaction time was not impacted by PART or DEP. In contrast the accuracy of responses was highly likely impaired by 2% (91) following DEP, while there was less certainty of impaired accuracy following PART (-1%, p = 73). Multiple aspects of physical and cognitive performance were impacted by sleep deprivation. The greatest detrimental effects were seen for aerobic performance and CMJ. Partial sleep deprivation equating to 4 hrs of sleep causes subtle, but potentially important negative impairments on athletic performance.

Low volume-high intensity interval exercise elicits antioxidant and anti-inflammatory effects in humans.

The purpose of the present study was to compare acute changes in oxidative stress and inflammation in response to steady state and low volume, high intensity interval exercise (LV-HIIE). Untrained healthy males (n = 10, mean ± s: age 22 ± 3 years; VO2MAX 42.7 ± 5.0 ml · kg(-1) · min(-1)) undertook three exercise bouts: a bout of LV-HIIE (10 × 1 min 90% VO2MAX intervals) and two energy-matched steady-state cycling bouts at a moderate (60% VO2MAX; 27 min, MOD) and high (80% VO2MAX; 20 min, HIGH) intensity on separate days. Markers of oxidative stress, inflammation and physiological stress were assessed before, at the end of exercise and 30 min post-exercise (post+30). At the end of all exercise bouts, significant changes in lipid hydroperoxides (LOOH) and protein carbonyls (PCs) (LOOH (nM): MOD +0.36; HIGH +3.09; LV-HIIE +5.51 and PC (nmol · mg(-1) protein): MOD -0.24; HIGH -0.11; LV-HIIE -0.37) were observed. Total antioxidant capacity (TAC) increased post+30, relative to the end of all exercise bouts (TAC (µM): MOD +189; HIGH +135; LV-HIIE +102). Interleukin (IL)-6 and IL-10 increased post+30 in HIGH and LV-HIIE only (P < 0.05). HIGH caused the greatest lymphocytosis, adrenaline and cardiovascular response (P < 0.05). At a reduced energy cost and physiological stress, LV-HIIE elicited similar cytokine and oxidative stress responses to HIGH.

The impact of intensified training with a high or moderate carbohydrate feeding strategy on resting and exercise-induced oxidative stress.

PURPOSE: This study investigated the impact of intensified training (IT) and carbohydrate (CHO) supplementation on resting and exercise-induced oxidative stress. METHODS: Male cyclists (n = 13, mean ± SD: age 25 ± 6 years; [Formula: see text] 72 ± 5 ml/kg/min) undertook two 9 day periods of endurance-based IT. In a counter-balanced, crossover and double-blinded study design, participants completed IT whilst ingesting high (H-CHO) or moderate (M-CHO) CHO beverages before (H-CHO: 24 g vs. M-CHO: 2 g), during (H-CHO: 60 g/h vs. M-CHO: 20 g/h) and after training sessions (H-CHO: 44 g vs. M-CHO: 10 g). Participants completed fasted performance trials without CHO on days 2, 6 and 10. Blood samples were taken before and immediately after exercise to assess plasma oxidative stress. RESULTS: Resting thiol (-SH) and catalase (CAT) activities decreased following 6 days of IT, independent of CHO condition [-SH (µM oxidised NADPH): H-CHO-14.0 ± 18.8, M-CHO-20.4 ± 20.3 and CAT (nmol/min/ml): H-CHO 12.5 ± 12.5, M-CHO 6.0 ± 4.5; all p < 0.05]. Resting total antioxidant capacity (TAC) was reduced after IT in M-CHO. All exercise bouts elicited significant increases in CAT, TAC, protein carbonylation (PC) and lipid hydroperoxides (LOOH), independent of CHO condition (p < 0.05). The magnitude of increase in PC and LOOH was greater on days 6 and 10 compared to day 2 in both conditions. CONCLUSIONS: Short-term IT caused reductions in resting antioxidant capacity in trained cyclists. Exercise-induced increases in PC and LOOH were exaggerated as a result of IT; however, these responses were independent of carbohydrate intake before, during and after the preceding IT sessions.

Underlying inflammation has no impact on the oxidative stress response to acute mental stress.

INTRODUCTION: Mental stress is considered to be a trigger for acute myocardial infarction (MI), with inflammation thought to provide a mechanism. Inflammation is reciprocally linked to oxidative stress, which has also been implicated in MI. The purpose of this study was to assess the effects of experimentally-induced inflammation on the oxidative stress response to mental stress in healthy participants. METHODS: Healthy males undertook one of two inflammatory stimuli: typhoid vaccination (Vaccination paradigm, N=17) or eccentric exercise (Eccentric exercise paradigm, N=17). All participants completed a mental arithmetic stress task twice (within-subject design): 6h after the inflammatory stimulus, and during a control non-inflammation condition. Blood samples were taken before, immediately and 30min after the stress task. Plasma was assessed for interleukin-6 (IL-6), protein carbonyls (PC), lipid hydroperoxides (LOOH), total antioxidant capacity (TAC) and nitric oxide metabolites (NOx). RESULTS: Vaccination paradigm: IL-6, PC and NOx were significantly higher in the vaccination condition, relative to the control condition (p<.05). PC, TAC, LOOH and NOx were unchanged in response to mental stress in both the vaccination and control conditions. Eccentric Exercise paradigm: IL-6 and TAC were significantly higher in the eccentric exercise condition (p<.05), relative to the control condition. PC, TAC and NOx were unchanged in response to mental stress in both the eccentric exercise and control conditions. CONCLUSIONS: Two different inflammatory paradigms were successful in increasing selective plasma markers of inflammation and oxidative stress prior to a mental stress task. However, experimentally induced transient inflammation had no impact on mental stress-induced changes in plasma LOOH, PC, TAC or NOx in young healthy participants.

Three months of moderate-intensity exercise reduced plasma 3-nitrotyrosine in rheumatoid arthritis patients.

PURPOSE: Rheumatoid arthritis (RA) patients display high levels of oxidative stress. Transient exercise-induced increases in oxidative stress are thought to be adaptive in healthy populations. This study investigated the effect of exercise on markers of oxidative stress in RA, following acute exercise and a period of exercise training. METHODS: Acute exercise study: RA patients (N = 12, age: 56 ± 11) undertook a bout of exercise (30-40 min, 70 % VO2MAX), and blood samples were taken before and after exercise to assess markers of oxidative stress. Training study: RA patients (N = 19, age: 56 ± 10) were randomised into either a control or exercise group, who undertook 3 exercise sessions per week (30-40 min @70 % VO2MAX) for 3 months. Plasma markers of oxidative stress (protein carbonyls (PC), lipid hydroperoxides (LOOH), 3-nitrotyrosine (3-NT), total antioxidant capacity (TAC) and catalase (CAT) activity), inflammation (interleukin-8 (IL-8) and C-reactive protein (CRP)) and nitric oxide metabolites (NOx) were assessed before and after training. RESULTS: Acute exercise study: Protein carbonyls (PC) (+18 %) and NOx (+27 %) were significantly increased following exercise. Training study: 3-nitrotyrosine (3-NT) decreased (2.18 ± 1.78 to 1.10 ± 0.93 µM) in the exercise group only, alongside increases in aerobic fitness (24.45 ± 4.98 to 27.10 ± 4.51 ml/kg/min(-1)) and reductions in disease activity score (DAS: 3.47 ± 1.17 to 2.88 ± 0.76). PC, LOOH, TAC, IL-8, CRP and NOx concentrations, and CAT activity were unchanged in both groups. CONCLUSIONS: Aerobic exercise training did not increase markers of oxidative stress in RA patients. 3-Nitrotyrosine and disease activity were decreased following exercise training.

Plasma concentrations of thioredoxin, thioredoxin reductase and peroxiredoxin-4 can identify high risk patients and predict outcome in patients with acute coronary syndrome: A clinical observation.

BACKGROUND: Oxidative stress is a pathological feature of acute coronary syndrome (ACS), a complex disease with varying clinical outcomes. Surrogate biomarkers of oxidative stress including, peroxiredoxin-2 (PRDX2), PRDX4, thioredoxin (TRX) and thioredoxin reductase (TRXR) were measured in ACS patients at presentation and follow-up, to assess their clinical utility in diagnosis and risk stratification. METHODS: Plasma from 145 participants (80 ACS and 65 healthy) at diagnosis, 1-3 month (first) and 6-month follow-up (second) was analysed by ELISA. ACS patients were monitored for 12-months. RESULTS: ACS patients at diagnosis had significantly higher concentrations of TRX (p < 0.05), TRXR (p < 0.01) and PRDX4 (p < 0.01), compared to healthy donors. This was increase was driven by non-ST elevated myocardial infarction for TRX (p < 0.01) and PRDX4 (p < 0.05). For TRXR, ACS females were significantly higher than males (p < 0.05). TRX was also higher in older females (>55 years) at diagnosis (p < 0.05). At first follow-up, TRX had lowered, whereas PRDX4 remained significantly high (p < 0.05). Stratification of ACS patients according to percutaneous coronary intervention (PCI) revealed that TRXR was significantly higher in patients receiving PCI to the right coronary artery (p < 0.05). Whereas both TRXR (p < 0.01) and PRDX4 (p < 0.01) were significantly higher in patients receiving PCI to the left anterior descending (LAD) artery. ACS patients who had plasma TRX >13.40 ng/ml at second follow-up were at high risk of readmission (p < 0.05), as were patients with TRXR of <1000 pg/ml at diagnosis having PCI to the LAD (p < 0.05). CONCLUSION: This study indicates that TRX, TRXR and PRDX4 may have clinical utility for ACS stratification.

Eccentric-exercise induced inflammation attenuates the vascular responses to mental stress.

Mental stress has been identified as a trigger of myocardial infarction (MI), with inflammation and vascular responses to mental stress independently implicated as contributing factors. This study examined whether inflammation moderates the vascular responses to mental stress. Eighteen healthy male participants completed a stress task under two counter balanced conditions. In the exercise condition, a morning bout of eccentric exercise (12×5 repetitions of unilateral eccentric knee extension at 120% intensity of concentric one repetition maximum) was used to increase levels of inflammatory-responsive cytokines during an afternoon stress session scheduled 6h later. In the control condition, participants sat and relaxed for 45min, 6h prior to the afternoon stress session. Forearm blood flow, calf blood flow (measured in the leg which completed the exercise task), blood pressure, heart rate and cardiac output were assessed at rest and in response to mental stress. As expected, interleukin-6 was higher (p=.02) 6h post exercise, i.e., at the start of the stress session, as compared to the no-exercise control condition. Mental stress increased forearm blood flow, calf blood flow, blood pressure, heart rate, and cardiac output in both conditions (p's<.001). Stress-induced calf blood flow was attenuated in the exercise condition compared to the control condition (p<.05) which was not the case for forearm blood flow. This study found that the inflammatory response to eccentric exercise attenuated the vascular responses to mental stress locally at the site of eccentric exercise-induced inflammation. The observed impairment in vascular responses to stress associated with increased levels of inflammation suggests a mechanism through which inflammation might increase the risk for MI.

Inactivity and obesity: consequences for macrophage-mediated inflammation and the development of cardiometabolic disease.

Obesity and dyslipidaemia are strongly associated with the development of cardiometabolic diseases including CVD, stroke, type 2 diabetes, insulin resistance and non-alcoholic fatty liver disease. While these conditions are preventable, they are leading causes of mortality globally. There is now overwhelming clinical and experimental evidence that these conditions are driven by chronic systemic inflammation, with a growing body of data suggesting that this can be regulated by increasing levels of physical activity and reducing sedentary time. In this review we address the role of macrophage-mediated inflammation on the development of cardiometabolic diseases in individuals with overweight and obesity and how reducing sedentary behaviour and increasing physical activity appears to lessen these pro-inflammatory processes, reducing the risk of developing cardiometabolic diseases. While loss of subcutaneous and visceral fat mass is important for reducing chronic systemic inflammation, the mediating effects of increasing physical activity levels and lowering sedentary time on the development of inflamed adipose tissue also occur independently of changes in adiposity. The message that weight loss is not necessary for the benefits of physical activity in lowering chronic inflammation and improving health should encourage those for whom losing weight is difficult. Additionally, while the health benefits of meeting the recommended physical activity guidelines are clear, simply moving more appears to lower chronic systemic inflammation. Reducing sitting time and increasing light physical activity may therefore provide an alternative, more approachable manner for some with overweight and obesity to become more active, reduce chronic inflammation and improve cardiometabolic health.

Adipose inflammation: South Asian ethnicity and central obesity are independently associated with higher immune cell recruitment to adipose-specific media: A pilot study in men.

A South Asian (SA) cardiovascular phenomenon exists whereby SAs have excess burden of cardiovascular disease (CVD) despite having low prevalence of recognized CVD risk factors. The aim of the current study was to determine whether perturbations in monocyte biology contribute to this phenomenon via higher circulating cell numbers, a more pro-inflammatory phenotype, and higher transmigration and adhesion. Adhesion is linked to vascular inflammation whereas transmigration is linked to tissue inflammation. SA men with (N = 10; SAs with central obesity [CO-SA]) and without (N = 10; lean SA [LE-SA]) central obesity, plus White European counterparts (N = 10; white Europeans with central obesity [CO-WE], N = 10; lean white Europeans [LE-WE]) participated. An ex vivo assay mimicking blood flow dynamics coupled to flow cytometry determined the adhesion and transmigration of monocyte subsets toward chemokine-rich media cultured from pre-adipocytes (absolute responses). Migration and adhesion were also standardized for differences in numbers of circulating monocytes between participants (relative responses). Metabolic and inflammatory markers were assessed. SAs had higher absolute (but not relative) adhesion and migration of monocytes than WEs. Central obesity was associated with higher absolute and relative adhesion and migration of monocytes. SAs had higher concentrations of all monocyte subsets compared with WEs coinciding with adverse cardiovascular-inflammatory profiles. LE-SAs had similar monocyte concentrations, transmigration, and adhesion compared with CO-WEs, corresponding with similar cardiovascular-inflammatory profiles. The study provides novel evidence for higher monocyte counts associated with higher transmigration and adhesion in SA compared with WE men. Importantly, similar monocyte biology and cardiovascular-inflammatory profiles were seen in LE-SAs compared with CO-WEs, which may contribute to the higher risk of CVD at lower body mass index experienced by SAs.

Effect of Acute Walking on Endothelial Function and Postprandial Lipemia in South Asians and White Europeans.

INTRODUCTION: South Asians (SAs) have an elevated risk of cardiovascular disease (CVD) compared with White Europeans (WEs). Postprandial endothelial function (flow-mediated dilatation (FMD%)) in SA women and SA men with central obesity has not been investigated. Research in other populations has highlighted that a 1% higher FMD% is associated with a ~13% lower risk of future CVD events. We investigated whether FMD% and lipemia, two markers for CVD risk, were higher in SAs versus WEs, whether walking improved FMD% and lipemia, and if there were ethnic differences in the response. METHODS: Lean premenopausal women (study 1; 12 SA, 12 WE) and men with central obesity (study 2; 15 SA, 15 WE) completed two 2-d trials. On day 1, participants walked for 60 min at 60% of their peak oxygen uptake or rested. On day 2, participants rested and consumed two high-fat meals over 8 h. Repeated ultrasound assessments of endothelial function and venous blood samples for CVD risk markers were taken. RESULTS: Compared with WEs, SAs had lower postprandial FMD% (study 1, -1.32%; study 2, -0.54%) and higher postprandial triacylglycerol concentrations (study 1, 0.31 mmol·L -1 ·h -1 ; study 2, 0.55 mmol·L -1 ·h -1 ). Walking improved postprandial FMD% (study 1, 1.12%; study 2, 0.94%) and resulted in no significant change or small reductions in postprandial triacylglycerol concentrations (study 1, -0.01 mmol·L -1 ·h -1 ; study 2, -0.25 mmol·L -1 ·h -1 ). Exercise-induced changes in FMD% and triacylglycerol were consistent between ethnic groups. CONCLUSIONS: Walking mitigated the adverse postprandial effect of a high-fat diet on FMD% to a similar extent in SA and WE women and men, even with no/small improvements in triacylglycerol. This study highlights the importance of exercise to clinically improve FMD% in SAs and WEs.

Acute Running and Coronary Heart Disease Risk Markers in Male Cigarette Smokers and Nonsmokers: A Randomized Crossover Trial.

PURPOSE: Cigarette smoking is an independent risk factor for coronary heart disease and is associated with impaired postprandial metabolism. Acute exercise reduces postprandial lipemia and improves other coronary heart disease risk markers in nonsmokers. Less is known about responses in cigarette smokers. METHODS: Twelve male cigarette smokers (mean ± SD; age = 23 ± 4 yr, body mass index = 24.9 ± 3.0 kg·m-2) and 12 male nonsmokers (age = 24 ± 4 yr, body mass index = 24.1 ± 2.0 kg·m-2) completed two, 2-d conditions (control and exercise) in a randomized crossover design. On day 1, participants rested for 9 h (0800-1700) in both conditions except a 60-min treadmill run (65% ± 7% peak oxygen uptake, 2.87 ± 0.54 MJ) was completed between 6.5 and 7.5 h (1430-1530) in the exercise condition. On day 2 of both conditions, participants rested and consumed two high-fat meals over 8 h (0900-1700) during which 13 venous blood samples and nine resting arterial blood pressure measurements were collected. RESULTS: Smokers exhibited higher postprandial triacylglycerol and C-reactive protein than nonsmokers (main effect group effect size [Cohen's d] ≥ 0.94, P ≤ 0.034). Previous day running reduced postprandial triacylglycerol, insulin, and systolic and diastolic blood pressure (main effect condition d ≥ 0.28, P ≤ 0.044) and elevated postprandial nonesterified fatty acid and C-reactive protein (main effect condition d ≥ 0.41, P ≤ 0.044). Group-condition interactions were not apparent for any outcome across the total postprandial period (0-8 h; all P ≥ 0.089), but the exercise-induced reduction in postprandial triacylglycerol in the early postprandial period (0-4 h) was greater in nonsmokers than smokers (-21%, d = 0.43, vs -5%, d = 0.16, respectively; group-condition interaction P = 0.061). CONCLUSIONS: Acute moderate-intensity running reduced postprandial triacylglycerol, insulin, and resting arterial blood pressure the day after exercise in male cigarette smokers and nonsmokers. These findings highlight the ability of acute exercise to augment the postprandial metabolic health of cigarette smokers and nonsmokers.

Sleep Deprivation: Cytokine and Neuroendocrine Effects on Perception of Effort.

INTRODUCTION: An increased perception of effort and subjective fatigue are thought to be central to decreased exercise performance observed after disrupted sleep. However, there is limited understanding of mechanisms that underpin these phenomena. We investigated the role of interleukin-6 (IL-6), the soluble IL-6 receptor, and neuroendocrine factors (cortisol, adrenaline, noradrenaline, and brain-derived neurotropic factor) in mediating these responses at rest and during exercise. METHODS: In a randomized order, 10 healthy active men completed three experimental trials following different sleep conditions: a single night of sleep deprivation, partial sleep deprivation equivalent to 4 h of sleep, and normal sleep. The experimental sessions consisted of physiological and perceptual measurements of exercise intensity throughout 45-min moderate intensity and 15-min maximal effort cycling. Cytokine and neuroendocrine factors were assessed at rest and in response to exercise. RESULTS: Sleep deprivation resulted in increased resting IL-6, lower blood glucose, increased perceived fatigue and perception of effort, lower free-living energy expenditure, and reduced maximal exercise performance. In contrast, sleep deprivation did not alter physiological, cytokine, or neuroendocrine responses to exercise. Variations in the resting concentration of IL-6 were associated with lowered blood glucose, an increased perception of effort, and impaired exercise performance. Resting concentrations of cortisol, adrenaline, noradrenaline, and BNDF showed subtle interactions with specific aspects of mood status and performance but were not affected by sleep deprivation. There were minimal effects of partial sleep deprivation. CONCLUSIONS: These findings demonstrate that cytokine and neuroendocrine responses to exercise are not altered by sleep deprivation but that changes in the resting concentration of IL-6 may play a role in altered perception of effort in this context.

Short-term High-fat Overfeeding Does Not Induce NF-κB Inflammatory Signaling in Subcutaneous White Adipose Tissue.

CONTEXT: It is unclear how white adipose tissue (WAT) inflammatory signaling proteins respond during the early stages of overnutrition. OBJECTIVE: To investigate the effect of short-term, high-fat overfeeding on fasting abdominal subcutaneous WAT total content and phosphorylation of proteins involved in nuclear factor-κB (NF-κB) inflammatory signaling, systemic metabolic and inflammatory biomarkers. DESIGN: Individuals consumed a high-fat (65% total energy from total fat), high-energy (50% above estimated energy requirements) diet for 7 days. RESULTS: Fifteen participants (aged 27 ± 1 years; body mass index 24.4 ± 0.6 kg/m2) completed the study. Body mass increased following high-fat overfeeding (+1.2 ± 0.2 kg; P < 0.0001). However, total content and phosphorylation of proteins involved in NF-κB inflammatory signaling were unchanged following the intervention. Fasting serum glucose (+0.2 ± 0.0 mmol/L), total cholesterol (+0.4 ± 0.1 mmol/L), low-density lipoprotein cholesterol (+0.3 ± 0.1 mmol/L), high-density lipoprotein cholesterol (+0.2 ± 0.0 mmol/L), and lipopolysaccharide-binding protein (LBP; +4.7 ± 2.1 µg/mL) increased, whereas triacylglycerol concentrations (-0.2 ± 0.1 mmol/L) decreased following overfeeding (all P < 0.05). Systemic biomarkers (insulin, soluble cluster of differentiation 14 [CD14], C-reactive protein, interleukin-6, tumor necrosis factor-α and monocyte chemoattractant protein-1) and the proportion and concentration of circulating CD14+ monocytes were unaffected by overfeeding. CONCLUSION: Acute lipid oversupply did not impact on total content or phosphorylation of proteins involved in WAT NF-κB inflammatory signaling, despite modest weight gain and metabolic alterations. Systemic LBP, which is implicated in the progression of low-grade inflammation during the development of obesity, increased in response to a 7-day high-fat overfeeding period.

High intensity interval exercise increases the frequency of peripheral PD-1+ CD8+ central memory T-cells and soluble PD-L1 in humans.

Exercise can exert anti-inflammatory effects in an intensity-dependent manner; however, the mechanisms mediating these effects are continually being established. Programme Death Receptor-1 (PD-1) is a membrane bound receptor that maintains immune tolerance by dampening immune cell interactions, such as those mediated by cytotoxic T-cell lymphocytes (CD8+). The aim of this study was to characterise sub-populations of CD8+ T-cells with regards to their expression of PD-1 before and immediately after exercise. Interleukin (IL)-6, soluble PD-1 (sPD-1) and its ligand (sPD-L1) were also quantified in plasma. Eight individuals (mean â€‹± â€‹SD: age 29 â€‹± â€‹5 years; BMI 24.2 â€‹± â€‹3.4 â€‹kg â€‹m2; V ˙ O2max 44.5 â€‹± â€‹6.4 â€‹ml â€‹kg-1·min-1) undertook two time and energy-matched cycling bouts in a counterbalanced study design: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE). Both MOD and HIIE increased the number, but not the proportion of circulating CD8+ PD-1+ cells, with no differences between trials. Within the CD8+ PD-1+ pool, the expression of PD-1 increased on central memory cells following HIIE only (fold change: MOD 1.0 vs HIIE +1.4), as well the concentration of CD8+PD-1+ memory cells within the circulation (cells/uL: MOD -0.4 vs HIIE +5.8). This response composed a very small part of the exercise-induced CD8+ lymphocytosis (Pre-Ex: 0.38% to Post-Ex: 0.69%; p â€‹> â€‹0.05). sPD-L1 and IL-6 concentration increased in tandem following MOD and HIIE (r â€‹= â€‹0.57; P â€‹= â€‹0.021), with a reciprocal decline in sPD-1 observed. The current data demonstrate that PD-1+ CD8+ lymphocytes were mobilised following both MOD and HIIE. Both the number of central memory CD8+ T-cells expressing PD-1 and the expression level on these cells were increased following HIIE only. This intensity-dependent phenotypic response, in conjunction with increased circulatory sPD-L1 may represent an aspect of the anti-inflammatory response to exercise and warrants further investigation.

Impact of aerobic exercise and fatty acid supplementation on global and gene-specific DNA methylation.

Lifestyle interventions, including exercise and dietary supplementation, can modify DNA methylation and exert health benefits; however, the underlying mechanisms are poorly understood. Here we investigated the impact of acute aerobic exercise and the supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) and extra virgin olive oil (EVOO) on global and gene-specific (PPARGC1A, IL6 and TNF) DNA methylation, and DNMT mRNA expression in leukocytes of disease-free individuals. Eight trained male cyclists completed an exercise test before and after a four-week supplementation of n-3 PUFA and EVOO in a double-blind, randomised, repeated measures design. Exercise triggered global hypomethylation (Pre 79.2%; Post 78.7%; p = 0.008), alongside, hypomethylation (Pre 6.9%; Post 6.3%; p < 0.001) and increased mRNA expression of PPARGC1A (p < 0.001). Associations between PPARGC1A methylation and exercise performance were also detected. An interaction between supplement and trial was detected for a single CpG of IL6 indicating increased DNA methylation following n-3 PUFA and decreased methylation following EVOO (p = 0.038). Global and gene-specific DNA methylation associated with markers of inflammation and oxidative stress. The supplementation of EVOO reduced DNMT1 mRNA expression compared to n-3 PUFA supplementation (p = 0.048), whereas, DNMT3a (p = 0.018) and DNMT3b (p = 0.046) mRNA expression were decreased following exercise. In conclusion, we demonstrate that acute exercise and dietary supplementation of n-3 PUFAs and EVOO induce DNA methylation changes in leukocytes, potentially via the modulation of DNMT mRNA expression. Future studies are required to further elucidate the impact of lifestyle interventions on DNA methylation.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

"Beet" the cold: beetroot juice supplementation improves peripheral blood flow, endothelial function, and anti-inflammatory status in individuals with Raynaud's phenomenon.

Raynaud's phenomenon (RP) is characterized by recurrent transient peripheral vasospasm and lower nitric oxide (NO) bioavailability in the cold. We investigated the effect of nitrate-rich beetroot juice (BJ) supplementation on 1) NO-mediated vasodilation, 2) cutaneous vascular conductance (CVC) and skin temperature (Tsk) following local cooling, and 3) systemic anti-inflammatory status. Following baseline testing, 23 individuals with RP attended four times, in a double-blind, randomized crossover design, following acute and chronic (14 days) BJ and nitrate-depleted beetroot juice (NDBJ) supplementation. Peripheral Tsk and CVC were measured during and after mild hand and foot cooling, and during transdermal delivery of acetylcholine and sodium nitroprusside. Markers of anti-inflammatory status were also measured. Plasma nitrite concentration ([nitrite]) was increased in the BJ conditions (P < 0.001). Compared with the baseline visit, thumb CVC was greater following chronic-BJ (Δ2.0 flux/mmHg, P = 0.02) and chronic-NDBJ (Δ1.45 flux/mmHg, P = 0.01) supplementation; however, no changes in Tsk were observed (P > 0.05). Plasma [interleukin-10] was greater, pan endothelin and systolic and diastolic blood pressure (BP) were reduced, and forearm endothelial function was improved, by both BJ and NDBJ supplementation (P < 0.05). Acute and chronic BJ and NDBJ supplementation improved anti-inflammatory status, endothelial function and blood pressure (BP). CVC following cooling increased post chronic-BJ and chronic-NDBJ supplementation, but no effect on Tsk was observed. The key findings are that beetroot supplementation improves thumb blood flow, improves endothelial function and anti-inflammatory status, and reduces BP in people with Raynaud's.NEW & NOTEWORTHY This is the first study to examine the effect of dietary nitrate supplementation in individuals with Raynaud's phenomenon. The principal novel findings from this study were that both beetroot juice and nitrate-depleted beetroot juice 1) increased blood flow in the thumb following a cold challenge; 2) enhanced endothelium-dependent and -independent vasodilation in the forearm; 3) reduced systolic and diastolic blood pressure, and pan-endothelin concentration; and 4) improved inflammatory status in comparison to baseline.

Characterization of extracellular redox enzyme concentrations in response to exercise in humans.

Redox enzymes modulate intracellular redox balance and are secreted in response to cellular oxidative stress, potentially modulating systemic inflammation. Both aerobic and resistance exercise are known to cause acute systemic oxidative stress and inflammation; however, how redox enzyme concentrations alter in extracellular fluids following bouts of either type of exercise is unknown. Recreationally active men (n = 26, mean ± SD: age 28 ± 8 yr) took part in either: 1) two separate energy-matched cycling bouts: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE) or 2) an eccentric-based resistance exercise protocol (RES). Alterations in plasma (study 1) and serum (study 2) peroxiredoxin (PRDX)-2, PRDX-4, superoxide dismutase-3 (SOD3), thioredoxin (TRX-1), TRX-reductase and interleukin (IL)-6 were assessed before and at various timepoints after exercise. There was a significant increase in SOD3 (+1.5 ng/mL) and PRDX-4 (+5.9 ng/mL) concentration following HIIE only, peaking at 30- and 60-min post-exercise respectively. TRX-R decreased immediately and 60 min following HIIE (-7.3 ng/mL) and MOD (-8.6 ng/mL), respectively. In non-resistance trained men, no significant changes in redox enzyme concentrations were observed up to 48 h following RES, despite significant muscle damage. IL-6 concentration increased in response to all trials, however there was no significant relationship between absolute or exercise-induced changes in redox enzyme concentrations. These results collectively suggest that HIIE, but not MOD or RES increase the extracellular concentration of PRDX-4 and SOD3. Exercise-induced changes in redox enzyme concentrations do not appear to directly relate to systemic changes in IL-6 concentration.NEW & NOTEWORTHY Two studies were conducted to characterize changes in redox enzyme concentrations after single bouts of exercise to investigate the emerging association between extracellular redox enzymes and inflammation. We provide evidence that SOD3 and PRDX-4 concentration increased following high-intensity aerobic but not eccentric-based resistance exercise. Changes were not associated with IL-6. The results provide a platform to investigate the utility of SOD3 and PRDX-4 as biomarkers of oxidative stress following exercise.

Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: An optimised protocol for digital flow cytometers.

Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [1]). There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of 'reductive stress' in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [4]). It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on: •The adaption of a protocol initially described by Durand and Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures.•Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase (Hole et al. (2013) [5]).•Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF (Lin and Maecker (2018) [6]).